RNA plays many important roles in cellular function. In the Central Dogma of Biology, RNA serves as a transient carrier of genetic information and as the adapter molecule that reads the code. RNA catalyzes reactions and serves in post-transcriptional gene regulation, development, and immunity. RNA also plays roles in human disease, including Prader-Willi and myotonic dystrophy. Understanding and harnessing the power of RNA, e.g. with RNAi, antisense technology, or with therapeutic ribozymes, requires an understanding of the structures of these RNA sequences. RNAstructure is a software package for RNA secondary structure prediction and analysis. It is available at: http://rna.urmc.rochester.edu and has been downloaded by a total of over 11,000 users. The specific aims of this proposal include both development and maintenance for the RNAstructure package: 1) Develop enthalpy nearest neighbor parameters for RNA secondary structure formation to allow structure prediction and analysis at other temperatures. 2) Construct a web server to provide RNA secondary structure nearest neighbor parameters and offer tutorials on their use. 3) Add RNAML support to the existing software to improve interoperability with other software packages. 4) Port RNAstructure to Unix, Linux, and Macintosh OS X using the Java runtime library to build a user-interface. 5) Provide high quality Adobe Acrobat File (pdf) output directly from RNAstructure, so that predicted structures can be edited for publication. 6) Update RNAstructure for 64-bit Microsoft Windows Vista. 7) Provide source code for download under an open license. 8) Construct a web server for the OligoWalk algorithm for predicting equilibrium binding affinities to an RNA target. Web servers are highly popular because of there ease of use. 9) Offer an email reflector for communication by the RNAstructure user community. This work has broad implications for human health. The computer tools contained in RNAstructure are used by others to determine RNA sequences that are good drug targets. RNAstructure is also useful for the design of RNA-based Pharmaceuticals.